1a.Objectives (from AD-416)
Obj 1: Identify genes that show epigenetic dysregulation in obesity using a candidate-gene approach. Subobj 1A. Characterize developmental establishment of DNA methylation at hypothalamic genes known to affect food intake regulation. Subobj 1B. Compare DNA methylation and expression of these genes between lean and obese mice. Obj 2: Determine if methylation and expression of specific genes in hypothalamus and/or adipose tissue differ between lean and obese mice. Subobj 2A. Identify genomic loci in hypothalamus and adipose tissue at which epigenetic dysregulation is associated with obesity. Subobj 2B. Determine if interindividual epigenetic variation at these loci is found before the onset of obesity. Obj 3: Determine if maternal obesity and/or nutrition before and during pregnancy persistently alters epigenetic regulation in offspring hypothalamus or adipose tissue. Subobj 3A. Identify genomic loci at which epigenetic dysregulation is induced by maternal obesity. Subobj 3B. Determine if this induced epigenetic dysregulation can be prevented by altering maternal diet. Obj 4: Identify placental epigenetic mechanisms that affect fetal nutrition, growth and development. Subobj 4A. Use methylation-specific amplification in conjunction with MSAM of genomic DNA extracted from trophoblast of normal term placentas and androgenetic complete hydatidiform moles to identify novel imprinted genes and other epigenetically regulated genes in trophoblast that play a role in regulation of fetal nutrition. Subobj 4B. Analyze how NLRP7, which is mutated in women with biparental hydatidiform moles, contributes to imprinting in early embryo and placenta. NLRP7 protein, known to have a role in innate immunity, may be a link between environmental changes (such as suboptimal maternal nutrition) and imprinting alterations during development. Obj 5: Determine how programming of glucose intolerance, obesity, and epigenetic dysregulation of skeletal muscle-growth in mice is affected by maternal diet during development. Preliminary data show that offspring of mice exposed to maternal low-protein (MLP) diet have reduction in skeletal muscle mass at <1 yr of age and possibly glucose intolerance. Subobj 5A. Extend phenotypic studies on muscle development, glucose tolerance, growth and obesity to include maternal methyl-donor depleted (MDD) diets and to follow offspring exposed to MLP or MDD diets for up to 18 months. Subobj 5B. To find the molecular basis for the altered muscle phenotype after MLP diet, we will perform array-based gene expression and methylation profiling, as well as gene expression analysis and DNA methylation analysis of candidate genes in hindleg muscle-tissues from 21-d-old and 1-yr-old mice exposed to MLP diet. Obj. 6 Utilize genome-wide DNA methylation profiling to determine if epigenetic programming/reprogramming contribute to lineage-specific patterns of gene expression. Obj. 7 Develop a targeted knock-in mouse model.

1b.Approach (from AD-416)
Children's Nutrition Research Center scientists will use the agouti viable yellow (Avy) mouse model to study the effects of maternal obesity on the risk of obesity in the offspring. DNA methylation in two tissues is known to play important roles in body weight regulation, hypothalamus and adipose tissue. Scientists will study this by using both a candidate gene approach and a genome-wide DNA methylation profiling technique (MSAM). In human studies, our research team will use MSAM and commercially available methylation screening tools such as the Illumina Infinium methylation array to identify genomic regions of differential methylation in complete hydatidiform moles compared with normal term placentas. Also, to characterize the role of NLRP7 in genomic imprinting, we will use chromatin immunoprecipitation to assess interactions of NLRP7 with chromatin, identify DNA binding sites of NLRP7 by electromobility shift assays, and screen for NLRP7 protein binding partners using yeast two-hybrid assays. Lastly, previous studies in a mouse model of the effects of maternal low protein diet on skeletal muscle development in the offspring will be extended to include longer-term studies and additional types of early exposures.

3.Progress Report

Most of the candidate genes we selected did not show developmental changes in DNA methylation (a chemical modification of DNA) in the hypothalamus (a key brain region responsible for body weight regulation). We therefore used a DNA methylation microarray approach (MSAM) to identify more genes at which DNA methylation changes are related to postnatal hypothalamic maturation. We identified over a thousand genomic regions undergoing postnatal DNA methylation changes, and validated over 10 by an independent method.
We longitudinally measured body weight in a cohort of Avy/a mice, and found that average body weight actually peaks at age 140 d (P140) and, in a subset of Avy/a mice, declines by P180. This indicates that much of the variation in body weight and adiposity we previously observed at P180 is attributable to variable weight loss, potentially due to some pathology. (Avy mice are known to be susceptible to several types of cancer.)
We have collected hypothalamus from over 40 P21 mice. Rather than focusing on spontaneous interindividual variation in Avy mice (for the reasons detailed above), we have developed a model of early postnatal overnutrition that induces persistent changes in body weight regulation, and will characterize hypothalamic DNA methylation changes in this model.
We have collected P21 hypothalamus from over 10 litters of both a/a and Avy/a mice born to either lean (a/a) or obese (Avy/a) mothers. All of these mice were cross-fostered to a/a mothers, so they were exposed to maternal obesity only prenatally.
We have performed DNA methylation profiling on Illumina arrays on 6 AnCHM tissues (hydatidiform molar pregnancies with only paternally inherited DNA in their genome), as an alternative to MSAM. Data analysis is currently in progress.
We have extensively tested several new antibodies to the NLRP7 protein and have now found one that seems to work in cells that highly express NLRP7 (in a parallel project not funded by USDA using human ES cells). Doing this we found some evidence that NLRP7 may be cleaved by caspase. This is novel and being confirmed. We have done assays with several candidate interactors (using overexpressed protein and now endogenous protein) to find their binding to NLRP7 and are working on ChIP to see if NLRP7 binds DNA. Yeast-two hybrid assays have been performed, but faced some difficulty. We also did yeast-two hybrid screens to find novel NLRP7 interactors; one interesting protein needs further confirmation.
Breeding of mice on various diets, GTTs and tissue collection are in progress.
We have isolated RNA from liver at 1 year of age, and muscle (soleus) at p21 and 1 year of age and performed gene expression profiling. We are working on data analysis and confirmatory assays. A manuscript is in preparation to describe the results on liver.
The ADODR monitors activities for the project by routine site visits, and review of major purchases of supplies/equipment, use of SCA funds for foreign travel, and submission of grant applications by investigators funded through the SCA.

4.Accomplishments
1.
Development of a mouse model to understand how maternal obesity during fetal development promotes obesity in the next generation. Previous studies have shown that maternal obesity promotes obesity in the next generation; but exactly when maternal obesity affects development, however, is not known. Researchers at the Children's Nutrition Research Center in Houston, Texas, have shown using a mouse model that maternal obesity during fetal development promotes excess fat levels in her offspring, whereas maternal obesity during the lactation period actually leads to lower body weight in the offspring. This is an important accomplishment in that it provides a 'cleaner' model in which we can study the effects of maternal obesity on epigenetics (developmental establishment of gene regulatory mechanisms) in the hypothalamus, and focuses the relevant period of development.

2.
Gene expression profiling in mouse model liver. Nutritional protein deprivation early in life has an impact on the liver, but the long-term consequences are not known, especially if this may predispose individuals to metabolic disease. Researchers at the Children's Nutrition Research Center in Houston, Texas, have found several up- and down-regulated genes that play a role in DNA organizational structure. This will help researchers understand the mechanisms of long-term altered gene regulation after early exposure to protein deprivation.